CN102758018A - Method for researching photosynthesis through combination of chlorophyll fluorescence technique and protoplast system, and application thereof - Google Patents
Method for researching photosynthesis through combination of chlorophyll fluorescence technique and protoplast system, and application thereof Download PDFInfo
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Abstract
The invention discloses a method for researching photosynthesis through combination of a chlorophyll fluorescence technique and a protoplast system, and application thereof. Genes related to photosynthesis can be screened at high throughput quickly through instantly expressing or silencing one or more of genes in a protoplast, determining change of chlorophyll fluorescence of the protoplast of an arabidopsis mesophyll cell, and comparing an image thereof.
Description
Technical field
The present invention relates to a kind of scientific research and use method, particularly a kind ofly be used to study photosynthetic method.
Background technology
Growth and development of plant be unable to do without photosynthesis, and photosynthesis is the basic substance of all substance metabolisms of organic sphere and energy metabolism, and it comprises the complex process that a series of optical physicss, photochemistry and biological chemistry change.Help people more in depth to understand photosynthetic process to photosynthetic research, and then on purpose to plant, particularly farm crop carry out the transgenic processing, to improve photosynthetic efficient.
At present, photosynthetic gene function is participated in research, mainly obtained to express, interfere plant through stable conversion, or through two mutants, the cycle is long, and workload is big.This research method efficient is low, is difficult to filter out efficiently the gene relevant with photosynthesis.
Protoplastis is after vegetable cell is removed cell walls, the material of that part of biologically active that is wrapped in by cytoplasmic membrane.Along with multiple protoplastis separates enzyme, like the discovery of cellulase, hemicellulase, polygalacturonase, macerozyme, driselase etc., the highly active protoplastis of a large amount of preparations has developed into a kind of routine techniques under laboratory condition.Separated the protoplastis that obtains many vegetable materials.Yoo etc. have reported a kind of method (Yoo that utilizes cellulase R-10 and macerozyme R-10 to separate protoplastis; S.D.; Cho, Y.H., and Sheen; J. (2007). Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis. Nat Protoc 2,1565-1572.).Present stage,, grope enzymolysis time through optimizing the enzyme liquid system of enzymolysis Arabidopis thaliana material; The improvement protoplastis prepares the committed step in the process, such as: centrifugal, filter etc. and can make things convenient for and obtain to have very in a large number apace high light closes active protoplastis (Riazunnisa; K., Padmavathi, L.; Scheibe, R., and Raghavendra; A.S. (2007). Preparation of Arabidopsis mesophyll protoplasts with high rates of photosynthesis. Physiol Plantarum 129,679-686).
Protoplastis transient expression technology is to be transformation receptor with the protoplastis, through certain method foreign gene is imported in the protoplastis, makes foreign gene be able to quick expression, and can keep the technology of expression level in a short time.
Protoplastis transient expression technology and traditional stable conversion compared with techniques, main difference are that stable conversion transforms foreign gene in the entering plant materials, and are incorporated in the Plant Genome, so gene information can be hereditary; And in the protoplastis transient expression system, then be that foreign gene is building up on the vector plasmid, transforming back transient expression in protoplastis, unconformability is in Plant Genome, and also not producing can hereditary offspring.Present stage, although there has not been technical problem in traditional stable conversion, the stable conversion cycle is long; Need loaded down with trivial details work to identify transgenic plant; Workload is big, and exogenous gene expression instability and efficient are low, thereby has satisfied not the demand of the high-throughput functional analysis of goal gene.In addition, the two mutants plant of portion gene or mistake expression plant can suddenly change deadly, and this makes the research work of this genoid be restricted.With the protoplastis is the generation of the gene transient expression technology of transformation receptor, helps alleviating this demand, has become the important technical (Sheen, 2001) in the present molecular biology research.
The report the earliest of relevant protoplastis transient expression technology is 1969, and Aoki and Takebe utilize tobacco mesophyll cell protoplast and tobacco mosaic virus(TMV) successfully exogenous nucleic acid to be introduced protoplastis (Aoki and Takebe, 1969) for the first time.After this, multiple DNA instantaneous conversion method has appearred, such as; Polyoxyethylene glycol (PEG) conversion method, electricity swash conversion method, particle gun conversion method etc., make DNA instantaneous conversion efficient be greatly improved, on this basis; The protoplastis that can prepare the various plants material; Transform goal gene, the research assignment of genes gene mapping, gene function and signal path (Yoo et al., 2007; Zhang et al., 2011).And new along with some, economic responsive reporter genes as: GRD beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) are introduced into the protoplastis transient expression system; Can reflect the expression of exogenous gene level according to reporter gene activity; Therefore the protoplastis transient expression system also obtains using more widely (Sheen, 2001).
Arabidopis thaliana mesophyll cell protoplastis is widely used in gene transient expression research (Yoo et al., 2007), signal path research (Sheen, 2001), ionic channel research (Lemtiri-Chlieh and Berkowitz, 2004; Qi et al., 2004) and research (An et al., 2003 of aspect such as molecular biology; Baek et al., 2004; Lemtiri-Chlieh and Berkowitz, 2004; Kim et al., 2006).
Modulation chlorophyll fluorescence imaging system have measure fast, simple, reliable, sample grown is not had basically characteristics such as influence to live body and measuring process, be widely used in physiology, the ecological study of algae and other higher plants.As, be used to screen the two mutants that chlorophyll fluorescence parameters changes, carry out genetic breeding.Niyogi etc. have done the photosynthetic mutant choice work of Chlamydomonas reinhardtii according to the difference of NPQ parameter; They also use the same method, and are that material has been made screening operation with the Arabidopis thaliana, identify the two mutants that 13 NPQ change altogether, have screened xenthophylls cycle mutant body (Niyogi et al., 1997 of algae and Arabidopis thaliana; Niyogi et al., 1998).In addition, though modulation chlorophyll fluorescence imaging system also capable of using screening influences plant metabolism and photosynthetic two mutants (Barbagallo et al., 2003) is not participated in growth directly.Because modulation chlorophyll fluorescence imaging system can reflect the difference and the variation of chlorophyll fluorescence parameters intuitively through imaging mode; Not only can detect the horizontal heterogeneity of leaf photosynthesis; Can detect also that early stage naked eyes are sightless coerces damage, or even illustrate micromechanism of damage and regulation mechanism.There is report modulation chlorophyll fluorescence imaging system can be applied to the visual rationing (Ehlert and Hincha, 2008) of freeze injury influence, and the survival time (Woo et al., 2008) after can qualitative assessment Arabidopis thaliana arid handling.Because modulation chlorophyll fluorescence imaging system imaging area is bigger, can measure a plurality of little algae samples simultaneously again, therefore also have bigger application (Escher et al., 2006) in the ecotoxicology field.Can detect the relevant physical signs of photosynthesis of plant intuitively, studying physiological process (Muller et al., 2001; Gould et al., 2010).
However, do not study photosynthetic method involving report so far through the chlorophyll fluorescence of measuring Arabidopis thaliana mesophyll cell protoplastis transient expression system.
Summary of the invention
The object of the present invention is to provide a kind of new photosynthesis research method.
The technical scheme that the present invention taked is:
A kind of method of studying photosynthesis correlation function gene comprises the steps:
1) preparation contains the protoplastis of chloroplast(id), but and testing gene and/or anti sense nucleotide sequence changed over to protoplastis and confirm that testing gene transient expression and/or anti sense nucleotide sequence have made the instantaneous silence of genetic expression;
The protoplastis that 2) will change testing gene and/or anti sense nucleotide sequence over to carries out the dark adatpation processing with the contrast protoplastis;
3) protoplastis of dark adatpation being handled carries out photo-irradiation treatment, obtains its fluoroscopic image and/or fluorescence parameter, or induces kinetic curve and/or photoinduction curve based on fluoroscopic image and/or fluorescence parameter drafting chlorophyll fluorescence;
4) fluoroscopic image, fluorescence parameter, the chlorophyll fluorescence of contrast different treatment group are induced at least a in kinetic curve, the photoinduction curve; Judge that testing gene and/or anti sense nucleotide sequence to photosynthetic influence, determine whether it is the photosynthesis genes involved.
The testing gene and/or the anti sense nucleotide sequence that change protoplastis over to are at least a kind of.
Preferably, the time of dark adatpation processing is 8～12 min.
Preferably, measuring chlorophyll fluorescence induces the program of kinetic curve and quick optical response curve to be:
1) opens deficiency so that protoplastis carries out photosynthetic faint measuring light, record minimum fluorescence F
0
2) with an after-applied saturation pulse, closeall electronic gate temporarily suppresses photosynthesis of plants, obtains maximum fluorescence Fm;
3) open actinic light, make protoplastis photosynthesis, apply saturation pulse after, the chlorophyll fluorescence that obtains is maximum fluorescence Fm ' under the actinic light;
4) treat that saturation pulse is handled after, the chlorophyll fluorescence value reaches after the stable state, closes actinic light, opens far-red light simultaneously, makes electronic gate get back to open attitude, F gets back to minimum fluorescence F
0Near, the fluorescence that obtain this moment is F
0'.
Preferably, protoplastis is an Arabidopis thaliana mesophyll cell protoplastis.
The invention has the beneficial effects as follows:
The inventive method is utilized protoplastis transient expression technology combined with fluorescent analytical technology, can the rapid determination testing gene to photosynthetic influence, help people more quickly and easily the photosynthesis genes involved to be studied.The inventive method can be studied a plurality of genes simultaneously, can carry out high flux screening.
Description of drawings
Fig. 1 is the microgram after the Arabidopis thaliana mesophyll cell protoplastis FDA dyeing;
Fig. 2 be around age wild-type with
Pgr5The chlorophyll fluorescence parameters comparison diagram of two mutants;
Fig. 3 is the photosynthetic electron transport inhibition design sketch of DCMU to leaf dish and protoplastis;
Fig. 4 is the fluorescence microscopy figure of the GFP of transient expression in the Arabidopis thaliana mesophyll cell protoplastis;
Fig. 5 is that instantaneous mistake is expressed the influence figure of NPQ-GFP to protoplastis;
Fig. 6 is that the instantaneous expression ATPC1-GFP that crosses is to protoplastis F
v/ F
mThe figure that influences with Ф PSII;
Fig. 7 is that the instantaneous expression ATPC1-c-myc that crosses is to protoplastis F
v/ F
mThe figure that influences with Ф PSII;
Fig. 8 is that DTT handles the figure that influences to the Ф PSII of protoplastis and blade and NPQ;
Fig. 9 is that instantaneous mistake is expressed TRX m1-GFP, TRX m2-GFP, TRX m3-GFP, the TRX m4-GFP figure that influences to the Ф PSII of protoplastis and NPQ;
Figure 10 is the instantaneous expression TRX f1-GFP that crosses, and TRX f2-GFP is to the figure that influences of the Ф PSII of protoplastis and NPQ;
Figure 11 is that instantaneous mistake is expressed the graph of a relation between TRX f1-GFP and the Ф PSII;
Figure 12 is the instantaneous expression Fd1-GFP that crosses, Fd2-GFP, and FTRc-GFP is to the figure that influences of protoplastis chlorophyll fluorescence parameters;
Figure 13 is Fd1-GFP, Fd2-GFP, FTRc-GFP, the Subcellular Localization figure of TRX f1-GFP and TRX f2-GFP.
Embodiment
Below in conjunction with test, further specify the present invention.
One, the protoplastis chlorophyll fluorescence is measured the foundation of system
Experiment material and solution:
The Arabidopis thaliana protoplastis prepares the compound method of solution
(1) mother liquor:
1. MES (0.2 M, pH 5.7): take by weighing 3.9 g MES and be dissolved in 100 mL deionized waters;
2. N.F,USP MANNITOL (0.8 M): take by weighing 29.1 g N.F,USP MANNITOL and be dissolved in 200 mL deionized waters;
3. CaCl
2(1 M): take by weighing 100 g CaCl
2Be dissolved in 100 mL deionized waters;
4. KCl (2 M): take by weighing 14.9 g KCl and be dissolved in 100 mL deionized waters;
5. MgCl
2(2 M): take by weighing 20.33 g MgCl
2Be dissolved in 100 mL deionized waters;
6. BSA (10 %): take by weighing 1 g BSA and be dissolved in 10 mL deionized waters, packing.
Above mother liquor is all used 0.45 μ m membrane filtration degerming, and normal temperature is preserved.
Enzyme liquid:
Proportioning is: 2 mL MES (0.2 M pH 5.7), 10 mL Mannitol (0.8 M), 200 μ L KCl (2 M), 0.3 g Cellulase R-10,0.08 g Macerozyme R-10,7.4 mL ddH
2O, 55 ℃ of heating 10 min, cool to room temperature adds 200 μ L CaCl again
2(1 M), 200 μ L BSA (10%).Mix, obtain with 0.45 μ m membrane filtration;
W5 solution:
Proportioning is: 2 mL MES (0.2 M pH 5.7), 1.8 g NaCl, 3.675 g CaCl
22H
2O, 0.5 mL KCl (2 M), 172.5 mL ddH
2O.
Vegetable material
The vegetable material that this test is used have the wild-type Arabidopis thaliana (
Arabidopsis thaliana, ecotype Columbia)
The cultivation of Arabidopis thaliana
(1) culture condition: temperature is 23 ℃, light intensity 110 μ molm
-2S
-1, 16 h illumination, 8 h are dark, relative humidity 60% ~ 70%.
(2) cultural method: get an amount of Arabidopis thaliana seed and place in 4 ℃ of refrigerators behind vernalization 4～5 d, take out seed, with its dibbling on the nutrition soil of the bacterium of going out.Cover translucent cover and cultivate, open cover behind 3 d with slow seedling.Treat seedling growth three to around the time, can use.
The preparation of Arabidopis thaliana mesophyll cell protoplastis
1) chooses 3 ~ 4 ages in week, wide-spread Arabidopsis leaf (leaf position: 2,3,4 pairs)
2) with blade blade is cut into the slice of 0.5 ~ 1 mm, puts into and be immersed in enzyme liquid;
3) enzyme liquid is vacuumized 30 min under the vacuum of 10 mm mercury column, the room temperature dark leaves standstill enzymolysis 3 h;
4) rock enzyme liquid and discharge protoplastis, add isopyknic W5 solution, rock plate gently, fully discharge protoplastis;
5) with the nylon net filter protoplastis of 55 μ m, be collected in the 15 mL round bottom plastic test tubes, centrifugal 1 ~ 2 min of 800 rpm, sedimentary protoplastis cleans once with the W5 solution of precooling, leaves standstill 30 min afterwards on ice.
The detection that Arabidopis thaliana mesophyll cell protoplastis prepares effect
(result is as shown in Figure 1 for fluorescein diacetate, FDA) staining detection protoplastis vigor to use fluorescein diacetate.Microscopically can be observed the protoplastis of making and is shaped as normal spherical shape, dyes behind 5 min with FDA, and blue light illumination is the activated protoplastis number of yellow-green colour near 100%.
The protoplastis chlorophyll fluorescence parameters is measured
The chlorophyll fluorescence that adopts the IMAGING-PAM-M-Series chlorophyll fluorescence appearance of German Walz company to measure protoplastis is induced kinetic curve and quick optical response curve.
When the mensuration chlorophyll fluorescence is induced kinetic curve, the protoplastis sample is added 96 orifice plates, dark adatpation 10 min earlier.Open software kit, selected interesting areas (AOI), the value of all fluorescence parameters is all got off through software records.With the low light level (0.5 μ molm
-2S
-1) irradiated with measurement initial fluorescence (F
0), modulating frequency is 1 Hz, carries out saturation pulse light (2 700 μ molm subsequently
-2S
-1) handle, after the pulse, obtain maximum fluorescence (Fm), open actinic light (the 35 μ molm that are fit to the Arabidopis thaliana protoplastis
-2S
-1), carry out the saturation pulse optical processing afterwards again, after the pulse, obtain the maximum fluorescence (Fm ') under the actinic light.
After the abundant dark adatpation, the maximum optical chemical efficiency (F of PSII
v/ F
m), to wait each parameter values all be that system calculates generation automatically under selected pattern for photochemistry energy the significant quantity suboutput [Y (II), Ф PSII], the non-photochemistry cancellation coefficient (NPQ) that transform.Image is directly derived observation, and each parameter value is further analyzed through related software after deriving.
Arabidopis thaliana mesophyll cell protoplastis chlorophyll fluorescence is measured choosing of plate
The high vigor wild-type Arabidopis thaliana mesophyll cell protoplastis of fresh separated is sub-packed in common transparent 96 orifice plates, measures its chlorophyll fluorescence parameters.
The result shows: the fluorescent signal in the transparent panel between each hole protoplastis can influence each other, and through attempting the different culture plate, finds that at last White-opalescent 96 orifice plates can avoid this influence effectively, and therefore, subsequent experimental is all used the White-opalescent plate.
Choosing of Arabidopis thaliana mesophyll cell protoplastis suspension matrix
Two kinds of suspension matrix of normal use in the Arabidopis thaliana mesophyll cell protoplastis transient expression system: W5 solution and WI solution.In order to select suitable suspension matrix, the preparation protoplastis suspends with W5 solution and WI solution respectively, places White-opalescent 96 orifice plates, measures chlorophyll fluorescence parameters F
v/ F
m
Experiment is found: than W5 solution, with the protoplastis that WI solution suspends, its F
v/ F
m(PSII maximum suboutput) is higher, is respectively: 0.72 and 0.68, and selected WI is a suspension matrix.
Different volumes, concentration Arabidopis thaliana mesophyll cell protoplastis F
v
/ F
m
Comparison
Preparation Arabidopis thaliana mesophyll cell protoplastis suspends with WI solution, and three volumes gradients (50 μ L, 100 μ L, 200 μ L), three concentration gradients (4 * 10 are set
5, 2 * 10
5, 1 * 10
5), protoplastis is placed White-opalescent 96 orifice plates respectively, measure chlorophyll fluorescence parameters F
v/ F
mExperiment is found: concentration is 4 * 10
5Individual/mL, the F of protoplastis when volume is 200 μ L
v/ F
mThe highest.Consider economic factors, selected volume, concentration system are: 2 * 10
5Individual/mL, volume is 100 μ L.
Two, the comparison of Arabidopis thaliana mesophyll cell protoplastis and leaf chlorophyll fluorescence parameter
Bibliographical information
Pgr5The NPQ of (PGR5 is the involved protein of ring type electron transport) two mutants is lower than wild-type (DalCorso et al., 2008), and weedicide dioxy phenyl dimethyl urea (DCMU) can be blocked the PSII electronics from Q
ATo secondary proton quinone acceptor (Q
B) transmit, cause Q
AReduction rapidly, the photochemistry cancellation is suppressed, initial fluorescence (F
0) increase (Allahverdiyeva et al., 2007).Therefore; Handle the leaf dish and the protoplastis of wild-type Arabidopis thaliana respectively with DCMU; Relatively DCMU handles the trend that the latter two chlorophyll fluorescence parameters change; Can confirm that the chlorophyll fluorescence parameters of protoplastis system and plant is whether similar or variation tendency is consistent, and then can definite chlorophyll fluorescence imaging technique be used to measure the chlorophyll fluorescence parameters of Arabidopis thaliana mesophyll cell protoplastis transient gene expression system.
Vegetable material:
The vegetable material that adopts is an Arabidopis thaliana homozygous mutation body
Pgr5, and the corresponding wild-type Arabidopis thaliana.The processing of Arabidopis thaliana and the preparation of protoplastis are the same.
The Arabidopsis leaf chlorophyll fluorescence parameters is measured
The chlorophyll fluorescence that adopts the Imaging-PAM-M-Series chlorophyll fluorescence appearance of German Walz company to measure Arabidopsis leaf is induced kinetic curve.
The Arabidopsis leaf chlorophyll fluorescence induces kinetic curve method for measuring and protoplastis chlorophyll fluorescence to induce the kinetic curve method for measuring similar, and difference is that the actinic light light intensity of suitable Arabidopsis leaf is 110 μ molm
-2S
-1
All around age wild-type,
Pgr5The NPQ of two mutants plant induces the measuring method of curve to be: 610 μ molm
-2S
-1High rayed 6 min of intensity, 4 min of dark adatpation subsequently;
All around age wild-type,
Pgr5The NPQ of two mutants protoplastis induces the measuring method of curve to be: 110 μ molm
-2S
-1High rayed 6 min of intensity, 4 min of dark adatpation subsequently.
Dioxy phenyl dimethyl urea (DCMU) is handled Arabidopis thaliana mesophyll cell protoplastis
Prepare 6 DCMU solution that gradient is 0,0.5,1,5,10,50 μ M, it is added respectively in 96 orifice plates, every hole 10 μ L.Subsequently, in each hole, add protoplastis 90 μ L, mix, the final concentration of DCMU in the protoplastis solution is respectively: 0,0.05,0.1,0.5,1,5 μ M.
DCMU handles Arabidopis thaliana leaf dish
Experimental result:
Whether the trend that changes for the chlorophyll fluorescence parameters of clear and definite protoplastis is consistent with blade, chooses
Pgr5And corresponding wild-type is as comparing.Measure the chlorophyll fluorescence parameters F of wild-type, two mutants blade earlier
v/ F
mWith NPQ.Subsequently, be that material prepares protoplasma respectively with them, measure the chlorophyll fluorescence parameters F of wild-type, two mutants protoplastis
v/ F
mWith NPQ.Experimental result is as shown in Figure 2, among the figure, around the A. age wild-type,
Pgr5Two mutants plant F separately
v/ F
m, 1/4 NPQ fluorescence imaging figure, the image of 1/4 NPQ is 110 μ molm
-2S
-1Intensity illuminated 3 min; B. around age wild-type,
Pgr5The NPQ of two mutants plant induces curve, 610 μ molm
-2S
-1High rayed 6 min of intensity, 4 min of dark adatpation subsequently, n=8; C. around age wild-type,
Pgr5Two mutants protoplastis F separately
v/ F
m, 1/4 NPQ fluorescence imaging figure, the image of 1/4 NPQ is 35 μ molm
-2S
-1Obtain behind intensity illuminated 3 min; D. around age wild-type,
Pgr5The NPQ of two mutants protoplastis induces curve, 110 μ molm
-2S
-1High rayed 6 min of intensity, 4 min of dark adatpation subsequently, n=6.
As can be seen from the figure, as far as blade, after the PGR5 sudden change, compare its non-photochemistry cancellation constant NPQ of wild-type and can reduce and F
v/ F
mRemarkable change (Fig. 2 A and B) but can not take place.The result is similar with blade, and the NPQ value of two mutants protoplastis is compared the wild-type protoplastis and significantly reduced (Fig. 2 C and D).
The comparison of chlorophyll fluorescence parameters behind DCMU processing wild-type Arabidopis thaliana leaf dish and the protoplastis
The leaf dish and corresponding protoplastis that prepare the wild-type Arabidopis thaliana respectively; Using concentration gradient is the leaf dish of the living type Arabidopis thaliana of DCMU processing of 0,0.5,1,5,10 and 50 μ M, and the use concentration gradient is that the DCMU of 0,0.05,0.1,0.5,1 and 5 μ M handles the protoplastis that the type of giving birth to Arabidopis thaliana prepares.
Experimental result is as shown in Figure 3, after A. DCMU handles among the figure, and the chlorophyll fluorescence parameters F of protoplastis (white post) and leaf dish (grey post)
v/ F
mFluorescence imaging figure and corresponding value thereof; B. after DCMU handles, the chlorophyll fluorescence parameters F of protoplastis (white post) and leaf dish (grey post)
0Fluorescence imaging figure and corresponding value thereof; C. after DCMU handles, the fluorescence imaging figure of chlorophyll fluorescence parameters 1/4 NPQ of protoplastis (white post) and leaf dish (grey post) and corresponding NPQ value thereof; D. after DCMU handles, the fluorescence imaging figure of the chlorophyll fluorescence parameters Ф PSII of protoplastis (white post) and leaf dish (grey post) and corresponding value thereof.Wherein, n=8.As shown in the figure, the used DCMU concentration of processing primary plastid is 0,0.05,0.1,0.5,1 and 5 μ M, and handling the used DCMU concentration of leaf dish then is 0,0.5,1,5,10 and 50 μ M.
Experimental result shows: along with the increase of DCMU concentration, and the initial fluorescence (F of leaf dish and protoplastis
0) all increase (Fig. 3 B) gradually, and F
v/ F
m, NPQ, the real-time fluorescence Ф PSII of PSII all can reduce gradually (Fig. 3 A, C, D).
Can know that through experiment the chlorophyll fluorescence parameters of protoplastis can reflect the truth of blade to a certain extent, can analyze the photosynthesis of blade through the chlorophyll fluorescence parameters of measuring protoplastis.
Three, in the Arabidopis thaliana protoplastis transient expression photosynthesis genes involved to the influence of its chlorophyll fluorescence parameters
In the Arabidopis thaliana mesophyll cell protoplastis transient expression technology, protoplastis need experience the DNA conversion process and the incubated overnight process of PEG mediation.Whether still be suitable for chlorophyll fluorescence mensuration in order clearly to transform the back protoplastis.Respectively in protoplastis instantaneous conversion GFP and NPQ1-GFP fusion rotein and GFP and ATPC1-GFP, c-myc and ATPC1-c-myc fusion rotein.Wherein, NPQ1 (Non-photochemical quenching 1), coding zeaxanthin diepoxide decyclization oxydase is as the key enzyme in the xenthophylls circulation; Under high light, can change zeaxanthin diepoxide into ZXN; Thereby dissipate superfluous luminous energy, play light protection (Niyogi et al., 1998).The arabidopsis mutant body of disappearance NPQ1, its NPQ can descend (Niyogi et al., 1998) greatly, can cause NPQ to increase (Havaux et al., 2000) and in tobacco, cross expression NPQ1.ATPC1 is a subunit of plastid atp synthase, the arabidopsis mutant body of disappearance ATPC1, and after the dark adatpation, its minimum fluorescence F
0Can increase, and, its F
v/ F
mCan significantly reduce (Bosco et al., 2004 with Ф PSII; Wu et al., 2007).
Vegetable material
This test used vegetable material be the wild-type Arabidopis thaliana (
Arabidopsis thaliana, ecotype Columbia Nossen), after the Arabidopis thaliana seed dried, places freezing preservation in 4 ℃ of refrigerators in 37 ℃ of incubators.
Arabidopis thaliana transforms the compound method of main solution
(1) WI solution (20 mL)
0.4?mL?MES（0.2?M?pH?5.7），12.5?mL?Mannitol（0.8?M），0.2?mL?KCl（2?M） 6.9?mL?ddH
2O
(2) MMG solution (10 mL)
0.2?mL?MES（0.2?M?pH?5.7），5?mL?Mannitol（0.8?M），0.15?mL?MgCl
2（1?M），4.65?mL?ddH
20：
(3) PEG/Ca
2+Solution (10 mL)
4?g?PEG?4000，2.5?mL?Mannitol（0.8?M），1?mL?CaCl
2（1?M），3?mL?ddH
2O。
The instantaneous conversion of Arabidopis thaliana mesophyll cell protoplastis
The resuspended protoplastis of MMG solution is quantitatively to 1-2 * 10
5Individual/mL
Add 10 μ L DNA (DNA 10 ~ 20 μ g of 5 kb size) at the round bottom EP of one 2 mL pipe, add 100 μ L protoplastiss (2 * 10
4Individual protoplastis), mixing;
Add 110 μ L PEG/Ca again
2+Solution, mixing;
Place behind 15 min with 440 μ L W5 solution dilutions the dark place, and mixing gently, and centrifugal 2 min of 800 rpm remove supernatant;
Add WI solution, disperse protoplastis, be tiled in overnight cultures in 6/12/24 orifice plate.
The affirmation of instantaneous conversion:
Extract the albumen that purifying transforms the back protoplastis, carry out protein electrophoresis and western blot, confirm whether the gene that is converted into protoplastis has expression.
In the Arabidopis thaliana protoplastis, behind the instantaneous conversion GFP carrier, through incubated overnight, collect protoplastis, covered on blood counting chamber sucks nucleonics from the side with protoplastis, under fluorescent microscope, observes.Add up transformation efficiency according to the protoplastis of green-emitting fluorescence and the ratio of all protoplastis numbers, observations is as shown in Figure 4.From figure, can know that the transformation efficiency of protoplastis can reach more than 90%.
The instantaneous mistake of NPQ1-GFP expressed back chlorophyll fluorescence parameters analysis
Instantaneous expression GFP and the NPQ1-GFP fusion rotein crossed in Arabidopis thaliana mesophyll cell protoplastis, incubated overnight is collected protoplastis; 800 rpm rotating speeds are centrifugal; The unnecessary nutrient solution of sucking-off with remaining culture liq and protoplastis mixing, is sub-packed in 96 orifice plates by every hole 100 μ L amount.Treat all samples packing completion, with measuring chlorophyll fluorescence parameters behind 96 hole dark adatpations, 10 min.Experimental result is as shown in Figure 5.Among the figure, the instantaneous mistake of A. protoplastis is expressed behind GFP, the NPQ1-GFP 1/4 NPQ fluorescence imaging figure separately, is six groups and repeats to get three groups; B. the instantaneous mistake of protoplastis is expressed measured separately NPQ numerical value behind GFP, the NPQ1-GFP, and numerical value is six groups and repeats to average * *
p≤0.01;
t-test; C. Western blot detects the GFP and the NPQ1-GFP albumen of transient expression; Last figure is that figure below is the painted pvdf membrane of CBB with the anti-immunoblotting of the multi-clone rabbit of anti-GFP, and star-like mark is represented non-specific band; RbcL is a ribulose-1,5-bisphosphate, the big subunit of 5-di-phosphate carboxylase.
Data show: cross the protoplastis of expressing the NPQ1-GFP fusion rotein, its NPQ was higher than the NPQ that expresses GFP albumen protoplastis.Numerical value be respectively 0.37 and 0.21 (Fig. 5 A, 5B).
Collect the protoplastis of having measured chlorophyll fluorescence parameters, extract albumen, carry out protein electrophoresis and western blot and detect.Western blot result shows: can detect the band of GFP and NPQ1-GFP fusion rotein correspondence with the antibody of anti-GFP, explain they can be in Arabidopis thaliana mesophyll cell protoplastis successful expression (Fig. 5 C).
These results show: protoplastis still has photosynthetic activity and physiological function through instantaneous conversion, is suitable for chlorophyll fluorescence parameters and measures.
The instantaneous mistake of ATPC1-GFP expressed back chlorophyll fluorescence parameters analysis
In order to prove that more fully the instantaneous protoplasma physical efficiency of crossing expression alien gene is used for chlorophyll fluorescence parameters and measures, choose that another had reported, it stablized the gene that chlorophyll fluorescence parameters of expressing plant can change
ATPC1Experimentize.Elder generation is instantaneous expression GFP and the ATPC1-GFP fusion rotein crossed in Arabidopis thaliana mesophyll cell protoplastis, and incubated overnight is collected protoplastis; 800 rpm rotating speeds are centrifugal; The unnecessary nutrient solution of sucking-off with remaining culture liq and protoplastis mixing, is sub-packed in 96 orifice plates by every hole 100 μ L amount.Treat all samples packing completion, with measuring chlorophyll fluorescence parameters behind 96 hole dark adatpations, 10 min.Experimental result is as shown in Figure 6.Among the figure, the A. protoplastis is instantaneous crosses behind expression GFP, the ATPC1-GFP F separately
v/ F
mFluorescence imaging figure and value corresponding; B. the instantaneous mistake of protoplastis is expressed behind GFP, the ATPC1-GFP fluorescence imaging figure and the value corresponding of Ф PSII separately; C. Western blot detects the GFP and the ATPC1-GFP albumen of transient expression, and upper part is that lower part is the painted pvdf membrane of CBB with the anti-immunoblotting of the multi-clone rabbit of anti-GFP, and RbcL is a ribulose-1,5-bisphosphate, the big subunit of 5-di-phosphate carboxylase; D. instantaneous fluorescence imaging figure and the value corresponding of Ф PSII separately crossed behind expression GFP (20 μ g), the ATPC1-GFP (0,5,10,15,20 μ g) of protoplastis; Fluorescence imaging figure gets three groups in six groups of repetitions among A, the B, and corresponding numerical value is six groups to be repeated to average; Fluorescence imaging figure is four groups and repeats to get one group among the D, and corresponding numerical value is four groups to be repeated to average; *
p≤0.05, * *
p≤0.01;
t-test.
Relatively cross the protoplastis of expressing the ATPC1-GFP fusion rotein and cross the protoplastis of expressing GFP.The former F
v/ F
mAll be higher than the latter with Ф PSII, numerical value is respectively F
v/ F
m(0.67 and 0.58) (Fig. 6 A), Ф PSII (0.35 and 0.28) (Fig. 6 B).
After proving instantaneous conversion ATPC1-GFP, protoplastis chlorophyll fluorescence parameters Fv/Fm and Ф PSII increase to be crossed to express by ATPC1 and cause, will
35S:ATPC1-GFPThe amount of fusion plasmid DNA transforms protoplastis after a gradient is set.In 100 μ L reaction systems, when
35S:ATPC1-GFPAmount when being lower than 10 μ g, transformation efficiency is very low, promptly transforms 20 μ g with contrast
35S:GFPThe protoplastis of plasmid is compared, and their Ф PSII is consistent (Fig. 6 D) almost.When transforming 15 or 20 μ g plasmids, the Ф PSII of protoplastis is significantly higher than contrast (Fig. 6 D).And the numerical value of Ф PSII increase along with the increase of ATPC1-GFP fusion rotein amount (Fig. 6 D, E).
Collect the protoplastis of having measured chlorophyll fluorescence parameters, extract albumen, carry out protein electrophoresis and western blot and detect.Western blot result shows: with the anti-band that can detect GFP and ATPC1-GFP fusion rotein correspondence of the multi-clone rabbit of anti-GFP, explain they can be in Arabidopis thaliana mesophyll cell protoplastis successful expression (Fig. 6 C).
Above result shows: instantaneous mistake is expressed its chlorophyll fluorescence parameters of protoplastis F of ATPC1 fusion rotein
v/ F
m, Ф PSII changes is crossed by ATPC1 albumen really that expression causes.
The instantaneous mistake of ATPC1-c-myc expressed back chlorophyll fluorescence parameters analysis
Understand the function of scattering and disappearing after considering some albumen and GFP amalgamation and expression, in order to reduce this influence as far as possible, direct sometimes expressing protein, or with albumen and less label amalgamation and expression, as: HA, c-myc, 6 * His, FLAG etc.
The reason that changes for protoplastis chlorophyll fluorescence parameters during further the last branch of proof is tested is the ATPC1 gene overexpression really.ATPC1 is connected among the transformed pUC-c-myc; Carrier construction ATPC1-c-myc, selecting the ecotype is the wild-type Arabidopis thaliana of Col-0 and No-0, the preparation protoplastis; Instantaneous mistake is expressed ATPC1 and 10 ATPC1-c-my that amino acid whose polypeptide merges, and c-myc is as contrast in instantaneous conversion simultaneously.
Experimental result is as shown in Figure 7.A.No-0 among the figure, the environmental Arabidopis thaliana protoplastis of Col-0 is instantaneous crosses behind expression c-myc, the ATPC1-c-myc F separately
v/ F
mFluorescence imaging figure and value corresponding; B. No-0, the instantaneous mistake of the environmental Arabidopis thaliana protoplastis of Col-0 is expressed behind c-myc, the ATPC1-c-myc fluorescence imaging figure and the value corresponding of Ф PSII separately; A-B, fluorescence imaging figure are six groups and repeat to get two groups that value corresponding is six groups and repeats to average * *
p≤0.01;
t-test.
Experimental result shows: in the protoplastis of these two environmental Arabidopis thalianas, behind the instantaneous expression ATPC1-c-myc excessively, compare photograph, the F of protoplastis
v/ F
m(Fig. 7 A, 7B), the protoplastis of expressing ATPC1-GFP with the instantaneous mistake of conversion is consistent all to compare the photograph height with Ф PSII.
Four, the rapid screening of photosynthesis correlation function gene
Report DTT according to existing research handles the leaf dish, can make its NPQ significantly descend (Bilger et al., 1989; Kalituho et al., 2007).DTT solution (0 ~ 10 mM) with concentration increases progressively is handled Arabidopis thaliana leaf dish and protoplastis, has also obtained consistent result.Because DTT is a kind of reduced form material, and can open proteinic disulfide linkage, the NPQ of inference Arabidopis thaliana, the regulation and control of the oxidated reductive action of Ф PSII possibility, further inference NPQ, Ф PSII may receive the regulation and control of chloroplast(id) redox system.
Trx (TRXs) is divided into 7 groups of (2f, 11h, 4m, 2o in the Arabidopis thaliana chloroplast(id) redox system; 1x, 2y and 1z), wherein 5 groups: 4m, 2f; 1x, 2y and 1z are considered to the localized Trx of plastid/chloroplast(id) (Gelhaye et al., 2005; Meyer et al., 2005; Arsova et al., 2010).The Trx that plays main regulating effect in the chloroplast(id) is TRX m and f, and they receive reduced form ferredoxin (Fd) to activate (Gelhaye et al., 2005 through receiving ferredoxin thioredoxin reductase (FTR) mediation; Meyer et al., 2005; Schurmann and Buchanan, 2008; Arsova et al., 2010).
Instantaneous mistake has been expressed 4 TRX m and 2 TRX f in protoplastis, measures chlorophyll fluorescence parameters.And then examination the FTR and the Fd at its upper reaches, study the redox regulation and control whether these genes participate in photosynthesis photoresponse center.
Experimental technique
DTT handles Arabidopis thaliana mesophyll cell protoplastis
Prepare 6 DTT solution that gradient is 0,2,10,20,40,100 mM, it is added respectively in 96 orifice plates, every hole 10 μ L.
Subsequently, in each hole, add protoplastis 90 μ L, mix, the final concentration of DTT in the protoplastis solution is respectively: 0,0.2,1,2,4,10 mM.
DTT handles Arabidopis thaliana leaf dish
After handling, with the residual DTT of deionized water flush away leaf panel surface, on the measurement plate of Imaging-PAM, complete the leaf dish, dark adatpation 10 min measure chlorophyll fluorescence.
Laser scanning co-focusing microscope is taken
Goal gene behind the transient expression, is collected protoplastis in protoplastis, (Leica TCS SP5 AOBS) observes with laser scanning co-focusing microscope, and shows and output image with Leica Microsystem LAS AF software.
GFP and chlorophyll autofluorescence (chl) use the laser excitation of 488 nm wavelength, use 500-530 nm to collect the GFP signal, collect the chlorophyll autofluorescence with 650-750 nm.
All independent at least triplicates of Fluirescence observation experiment.
DTT handles the comparison of chlorophyll fluorescence parameters behind wild-type Arabidopis thaliana leaf dish and the protoplastis respectively
The DTT solution of 6 concentration gradients of preparation is respectively: 0,0.2,1,2,4,10 mM.
Arabidopis thaliana leaf dish is immersed in the DTT solution behind 20 min, takes out the leaf dish, the DTT of water flush away leaf panel surface.Measure chlorophyll fluorescence parameters after the dark adatpation; Experimental result is as shown in Figure 8; Among the figure, A is with fluorescence imaging figure and the value corresponding of the measured chlorophyll fluorescence parameters Ф PSII of the DTT solution-treated Arabidopis thaliana leaf dish of different concns (0,0.2,1,2,4 and, 10 mM); Fluorescence imaging figure and the value corresponding of B 1/4 NPQ; C is with fluorescence imaging figure and the value corresponding of the measured chlorophyll fluorescence parameters Ф PSII of the DTT solution-treated Arabidopis thaliana mesophyll cell protoplastis of different concns (0,0.2,1,2,4 and, 10 mM); Fluorescence imaging figure and the value corresponding of D 1/4 NPQ; Fluorescence imaging figure is six groups and repeats to get one group that value corresponding is six groups and repeats to average.
The NPQ meeting of being found the leaf dish by experimental data significantly reduces, and significantly rising of Ф PSII (Fig. 8 A, B).
Survey chlorophyll fluorescence parameters behind the DTT processing primary plastid with same concentration gradient, find that the NPQ of protoplastis also can reduce along with the rising of DTT concentration, Ф PSII then raise along with the rising of DTT concentration (Fig. 8 C, D).
TRX m1, TRX m2, TRX m3, the instantaneous mistake of TRX m4-GFP are expressed back chlorophyll fluorescence parameters analysis
Whether participate in NPQ and Ф PSII in order to study the Trx that plays main regulating effect in the Arabidopis thaliana chloroplast(id); Trx (the TRX m1-GFP that instantaneous mistake has been expressed 4 m classes in Arabidopis thaliana mesophyll cell protoplastis respectively earlier; TRX m2-GFP; TRX m3-GFP and TRX m4-GFP), measured the chlorophyll fluorescence of expressing protoplastis, detect them and whether can cause the chlorophyll fluorescence parameters Ф PSII and the NPQ of protoplastis to change.Experimental result is as shown in Figure 9, A. is instantaneous among the figure cross expression GFP, TRX m1-GFP, TRX m2-GFP, TRX m3-GFP, TRX m4-GFP after, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters Ф PSII; B. behind instantaneous expression GFP, TRX m1-GFP, TRX m2-GFP, TRX m3-GFP, the TRX m4-GFP excessively, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters NPQ; A-B, fluorescence imaging figure are six groups and repeat to get three groups that value corresponding is six groups and repeats to average.
Repeat through biology more than 6 times, experimental result shows: although can detect the fluorescence behind 4 m similar thioredoxins and the GFP expressing fusion protein through fluorescent microscope, that is to say that these four albumen can express in protoplastis.But, they in protoplastis respectively instantaneous cross express after equal remarkable chlorophyll fluorescence parameters (Fig. 9) of change protoplastis not.
TRX f1, the instantaneous mistake of TRX f2-GFP are expressed back chlorophyll fluorescence parameters analysis
Instantaneous mistake is expressed the chlorophyll fluorescence parameters of measuring protoplastis behind the m similar thioredoxin, finds that they are crossed to express the back chlorophyll fluorescence parameters and do not take place significantly to change.On this basis; Whether can influence the chlorophyll fluorescence parameters Ф PSII and the NPQ of protoplastis through redoxomorphism for the Trx of understanding another kind of main Trx f class; Instantaneous mistake is expressed the Trx (TRX f1-GFP and TRX f2-GFP) of 2 f classes in Arabidopis thaliana mesophyll cell protoplastis, measures the chlorophyll fluorescence of expressing the back protoplastis.Experimental result is shown in figure 10, among the figure, and behind instantaneous expression GFP excessively of A. and the TRX f1-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters Ф PSII; B. behind instantaneous expression GFP excessively and the TRX f1-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters NPQ; C. behind instantaneous expression GFP excessively and the TRX f2-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters Ф PSII; D. behind instantaneous expression GFP excessively and the TRX f2-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters NPQ; E. Western blot detects the TRX f1-GFP of transient expression, TRX f2-GFP, GFP albumen; Upper part is with the anti-immunoblotting of the multi-clone rabbit of anti-GFP; Lower part is the painted pvdf membrane of CBB, and RbcL is a ribulose-1,5-bisphosphate, the big subunit of 5-di-phosphate carboxylase; A-D, fluorescence imaging figure are six groups and repeat to get three groups that value corresponding is six groups and repeats to average *
p≤0.05, * *
p≤0.01;
t-test.
The result shows, instantaneous cross expression TRX f1-GFP and TRX f2-GFP after, the trend that changes after protoplastis chlorophyll fluorescence parameters Ф PSII and NPQ and the DTT processing is consistent.Compared the contrast of expressing GFP, and crossed expression TRX f1-GFP, its Ф PSII of the protoplastis of TRX f2-GFP raises (Figure 10 A, C) to some extent, NPQ decrease (Figure 10 B, D).
In order to prove that protoplastis chlorophyll fluorescence parameters Ф PSII raises is to be crossed to express by TRX f to cause, at first will
35S:TRX f1-GFPThe amount of fusion plasmid DNA is provided with 4 gradients: 5,10,15 and 20 μ g transform protoplastis.
In 100 μ L protoplastis systems, when
35S:TRX f1-GFPAmount when being lower than 10 μ g, transformation efficiency is very low, can only detect the TRX f1-GFP albumen of very small amount, promptly transforms 20 μ g with contrast
35S:GFPThe protoplastis of plasmid is compared, and their Ф PSII is almost consistent.When transforming 15 or 20 μ g plasmids, the Ф PSII of protoplastis is significantly higher than contrast (Figure 11 A).
For Arabidopis thaliana mesophyll cell protoplastis transient expression system, in the system of 100 μ L protoplastiss, 20 μ g plasmids transform 2 * 10
4The changing effect of individual protoplastis is generally best.Cross expression and can cause protoplastis Ф PSII to increase in order further to confirm that TRX f is instantaneous in protoplastis, will
35S:TRX f1-GFPThe amount of fusion plasmid DNA is provided with other 4 gradients: 7.5,15,22.5 and 30 μ g transform protoplastis.Experimental result shows: transform the protoplastis of 22.5 μ g plasmids, its Ф PSII is the highest, transforms the protoplastis of 15 and 30 μ g plasmids, its Ф PSII all decrease (Figure 11 B).
Collect the protoplastis of having measured chlorophyll fluorescence parameters, extract albumen, carry out protein electrophoresis and western blot and detect.Western blot result shows: after anti-and corresponding two anti-the hatching with anti-GFP, can detect the band of GFP and TRX f1-GFP fusion rotein correspondence, explain they can be in Arabidopis thaliana mesophyll cell protoplastis successful expression.The result shows: for the protoplastis system of 100 μ L, the optimum amount that transforms plasmid is about 20 μ g, and this moment, the Expression of Fusion Protein amount was the highest, and the increase of plasmid amount or minimizing all can reduce transformation efficiency.And protoplastis chlorophyll fluorescence parameters Ф PSII can along with TRX f1-GFP fusion protein expression increase and increase (Figure 11 C, D).
Above result can illustrated together: instantaneous mistake expresses that its chlorophyll fluorescence parameters of protoplastis Ф PSII of TRX f1-GFP fusion rotein increases is crossed by TRX f1 albumen really that expression causes.
Fd1-GFP, Fd2-GFP, the instantaneous mistake of FTRc-GFP are expressed back chlorophyll fluorescence parameters analysis
Because TRX f is reduced by FTR, FTR that is to say that by Fd reduction (Schurmann and Buchanan, 2008) FTR and Fd are in the upper reaches of TRX f.Whether the albumen of expressing the TRX f upper reaches for clear and definite instantaneous mistake can cause that identical change takes place chlorophyll fluorescence parameters, has chosen the Fd:Fd1 and the Fd2 that express in two leaves, and the catalytic subunit FTRc of FTR, and their instantaneous mistakes in protoplastis are expressed.
Experimental result is shown in figure 12, among the figure, and the instantaneous expression GFP that crosses of A., behind FTRc-GFP and the TRX f2-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters Ф PSII; B. the instantaneous expression GFP that crosses, behind FTRc-GFP and the TRX f2-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters NPQ; C. the instantaneous expression GFP that crosses, Fd1-GFP, Fd2-GFP, behind TRX f1-GFP and the TRX f2-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters Ф PSII; D. the instantaneous expression GFP that crosses, Fd1-GFP, Fd2-GFP, behind TRX f1-GFP and the TRX f2-GFP, fluorescence imaging figure and the value corresponding thereof of protoplastis chlorophyll fluorescence parameters NPQ; E. Western blot detects the Fd1-GFP of transient expression, Fd2-GFP, FTRc-GFP; GFP albumen, upper part are that lower part is the painted pvdf membrane of CBB with the anti-immunoblotting of the multi-clone rabbit of anti-GFP; RbcL is a ribulose-1,5-bisphosphate, the big subunit of 5-di-phosphate carboxylase; A-D, fluorescence imaging figure are six groups and repeat to get two groups that value corresponding is six groups and repeats to average *
p≤0.05, * *
p≤0.01;
t-test.
The result shows: although cross expression TRX f1-GFP, and the protoplastis of TRX f2-GFP, its Ф PSII meeting is significantly risen, and NPQ is decline (Figure 12 A significantly; B, C, D); But do not find expression Fd1 and Fd2, and the chlorophyll fluorescence parameters of the protoplastis of FTRc, (Figure 12 A takes place to change significantly like NPQ or Ф PSII; B, C, D).Western-blot result shows, GFP, FTRc-GFP, Fd1-GFP and Fd2-GFP can be in protoplastis normal expression (Figure 12 E).
Explain that the f similar thioredoxin is through redoxomorphism direct regulation and control NPQ and Ф PSII; Because the f similar thioredoxin is more stable at the intravital content of plant; It is inoperative that instantaneous mistake is expressed the upstream gene of f similar thioredoxin; Also there is another kind of possibility, the function of promptly having scattered and disappeared behind these albumen and the GFP amalgamation and expression.However; Utilize novel method; Arabidopis thaliana mesophyll cell protoplastis instantaneous conversion technology is combined with the chlorophyll fluorescence technology, and results suggest TRX f1 that is obtained and TRX f2 possibly be two potential Trxs of redox regulation and control NPQ and Ф PSII.
The Subcellular Localization of Fd1, Fd2, FTRc, TRX f1, TRX f2
The reason that changes in order to ensure chlorophyll fluorescence parameters is that the instantaneous of foreign gene expressed excessively; Must there be the testing goal gene that the method for expressing has taken place to cross; Such as, through western blot, with antibody test goal gene and the GFP Expression of Fusion Protein of anti-GFP.Can also utilize the fluorescence of fluorescence microscope goal gene and GFP fusion rotein, instantaneous conversion TRX f1-GFP, TRX f2-GFP; FTRc-GFP, Fd1-GFP, Fd2-GFP; Can detect this 5 gene successful expression with laser confocal microscope, and be positioned at (Figure 13) in the chloroplast(id).
It is thus clear that the inventive method can be advantageously applied to photosynthetic research.
Based on above-mentioned experiment, can predict clearly, sense-rna is changed in the protoplastis, make the instantaneous silence of normal expression gene in the protoplastis, can be applied to photosynthetic research equally.
Claims (5)
1. a method of studying photosynthesis correlation function gene comprises the steps:
1) preparation contains the protoplastis of chloroplast(id), but and testing gene and/or anti sense nucleotide sequence changed over to protoplastis and confirm that testing gene transient expression and/or anti sense nucleotide sequence have made the instantaneous silence of genetic expression;
The protoplastis that 2) will change testing gene and/or anti sense nucleotide sequence over to carries out the dark adatpation processing with the contrast protoplastis;
3) protoplastis of dark adatpation being handled carries out photo-irradiation treatment, obtains its fluoroscopic image and/or fluorescence parameter, or induces kinetic curve and/or photoinduction curve based on fluoroscopic image and/or fluorescence parameter drafting chlorophyll fluorescence;
4) fluoroscopic image, fluorescence parameter, the chlorophyll fluorescence of contrast different treatment group are induced at least a in kinetic curve, the photoinduction curve; Judge that testing gene and/or anti sense nucleotide sequence to photosynthetic influence, determine whether it is the photosynthesis genes involved.
2. method according to claim 1 is characterized in that: the testing gene and/or the anti sense nucleotide sequence that change protoplastis over to are at least a kind of.
3. method according to claim 1 and 2 is characterized in that: the time that dark adatpation is handled is 8～12 min.
4. method according to claim 1 and 2 is characterized in that: measure chlorophyll fluorescence and induce the program of kinetic curve and quick optical response curve to be:
1) opens deficiency so that protoplastis carries out photosynthetic faint measuring light, record minimum fluorescence F
0
2) with an after-applied saturation pulse, closeall electronic gate temporarily suppresses photosynthesis of plants, obtains maximum fluorescence Fm;
3) open actinic light, make protoplastis photosynthesis, apply saturation pulse after, the chlorophyll fluorescence that obtains is maximum fluorescence Fm ' under the actinic light;
4) treat that saturation pulse is handled after, the chlorophyll fluorescence value reaches after the stable state, closes actinic light, opens far-red light simultaneously, makes electronic gate get back to open attitude, F gets back to minimum fluorescence F
0Near, the fluorescence that obtain this moment is F
0'.
5. method according to claim 1 and 2 is characterized in that: protoplastis is an Arabidopis thaliana mesophyll cell protoplastis.
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| CN105331574A (en) * | 2015-11-16 | 2016-02-17 | 华南农业大学 | Method for preparation and transient gene expression of jatropha curcas tissue culture seedling protoplast |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104303867A (en) * | 2014-10-16 | 2015-01-28 | 中国科学院遗传与发育生物学研究所 | Method for comparing hard-light resistance of plants |
| CN105331574A (en) * | 2015-11-16 | 2016-02-17 | 华南农业大学 | Method for preparation and transient gene expression of jatropha curcas tissue culture seedling protoplast |
| CN112903642A (en) * | 2021-01-20 | 2021-06-04 | 井冈山大学 | Method for quantitatively analyzing photosynthetic heterogeneity of plant leaves |
| CN112903642B (en) * | 2021-01-20 | 2022-06-10 | 井冈山大学 | Method for quantitatively analyzing photosynthetic heterogeneity of plant leaves |
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